Combining local regional therapy and systemic therapy: Expected changes in the treatment landscape of recurrent hepatocellular carcinoma.

Improvements in early screening, new diagnostic techniques, and surgical treatment have led to continuous downward trends in hepatocellular carcinoma (HCC) morbidity and mortality rates. However, high recurrence and refractory cancer after hepatectomy remain important factors affecting the long-term prognosis of HCC. The clinical characteristics and prognosis of recurrent HCC are heterogeneous, and guidelines on treatment strategies for recurrent HCC are lacking. Therapies such as surgical resection, radiofrequency ablation, and transhepatic arterial chemoembolization are effective for tumors confined to the liver, and targeted therapy is a very important treatment for unresectable recurrent HCC with systemic metastasis. With the deepening of the understanding of the immune microenvironment of HCC, blocking immune checkpoints to enhance the antitumor immune response has become a new direction for the treatment of HCC. In addition, improvements in the tumor immune microenvironment caused by local treatment may provide an opportunity to improve the therapeutic effect of HCC treatment. Ongoing and future clinical trial data of combined therapy may develop the new treatment scheme for recurrent HCC. This paper reviews the pattern of recurrent HCC and the characteristics of the immune microenvironment, demonstrates the basis for combining local treatment and systemic treatment, and reports current evidence to better understand current progress and future approaches in the treatment of recurrent HCC.

Dual targeting of Polo-like kinase 1 and baculoviral inhibitor of apoptosis repeat-containing 5 in TP53-mutated hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC), often diagnosed at advanced stages without curative therapies, is the fifth most common malignant cancer and the second leading cause of cancer-related mortality. Polo-like kinase 1 (PLK1) is activated in the late G2 phase of the cell cycle and is required for entry to mitosis. Interestingly, PLK1 is overexpressed in many HCC patients and is highly associated with poor clinical outcome. Baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5) is also highly overexpressed in HCC and plays key roles in this malignancy. AIM: To determine the expression patterns of PLK1 and BIRC5, as well as their correlation with p53 mutation status and patient clinical outcome. METHODS: The expression patterns of PLK1 and BIRC5, and their correlation with p53 mutation status or patient clinical outcome were analyzed using a TCGA HCC dataset. Cell viability, cell apoptosis, and cell cycle arrest assays were conducted to investigate the efficacy of the PLK1 inhibitors volasertib and GSK461364 and the BIRC5 inhibitor YM155, alone or in combination. The in vivo efficacy of volasertib and YM155, alone or in combination, was assessed in p53-mutated Huh7-derived xenograft models in immune-deficient NSIG mice. RESULTS: Our bioinformatics analysis using a TCGA HCC dataset revealed that PLK1 and BIRC5 were overexpressed in the same patient subset and their expression was highly correlated. The overexpression of both PLK1 and BIRC5 was more frequently detected in HCC with p53 mutations. High PLK1 or BIRC5 expression significantly correlated with poor clinical outcome. PLK1 inhibitors (volasertib and GSK461364) or a BIRC5 inhibitor (YM155) selectively targeted Huh7 cells with mutated p53, but not HepG2 cells with wild-type p53. The combination treatment of volasertib and YM155 synergistically inhibited the viability of Huh7 cells via apoptotic pathway. The efficacy of volasertib and YM155, alone or in combination, was validated in vivo in a Huh7-derived xenograft model. CONCLUSION: PLK1 and BIRC5 are highly co-expressed in p53-mutated HCC and inhibition of both PLK1 and BIRC5 synergistically compromises the viability of p53-mutated HCC cells in vitro and in vivo.

Hv1-deficiency protects ß cells from glucotoxicity through regulation of NOX4 level.

High glucose (HG)-induced oxidative stress contributes to the dysfunction of pancreatic ß cells in diabetes. The voltage-gated proton channel Hv1 has been proposed to support reactive oxygen species (ROS) production during respiratory bursts. However, the effect of Hv1 on glucotoxicity in pancreatic ß cells is not clear yet. In this study, we examined the protective effects of Hv1-deficiency in HG cultured ß cells. Following 48 h of treatment with 30 mM high glucose, Hv1 KO ß cells showed higher cell viability, lower cell apoptosis and a more stable insulin gene expression level compared to WT ß cells. In both control and HG cultured ß cells, deficiency of Hv1 decreased the glucose- and PMA-induced ROS production. Finally, HG incubation led to NOX4 upregulation in WT ß cells, which could be inhibited by HV1 deficiency. In conclusion, Hv1-deficiency prevents the HG treatment-induced NOX4 upregulation and protects ß cells from glucotoxicity.

Negative impact of hepatitis B surface seroclearance on prognosis of hepatitis B-related primary liver cancer.

AIM: To assess the impact of hepatitis B surface (HBsAg) seroclearance on survival outcomes in hepatitis B-related primary liver cancer. METHODS: Information from patients with hepatitis B-related liver cancer admitted in our hospital from 2008-2017 was retrieved. Cases diagnosed with HBsAg (-) and HBcAb (+) liver cancer were included in the HBsAg seroclearance (SC) group. HBsAg (+) liver cancer patients strictly matched for liver cancer stage (AJCC staging system, 8th edition), Child-Pugh score, and first diagnosis/treatment method (surgery, ablation and TACE) were assigned to the HBsAg non-seroclearance (NSC) group. Then, clinical, pathological and survival data in both groups were assessed. RESULTS: The SC and NSC groups comprised of 72 and 216 patients, respectively. Patient age (P < 0.001) and platelet count (P = 0.001) in the SC group were significantly higher than those of the NSC group. SC group patients who underwent surgery had more intrahepatic cholangiocarcinoma (ICC) and combined HCC-CC (CHC) cases than the NSC group, but no significant differences in tumor cell differentiation and history of liver cirrhosis were found between the two groups. The numbers of interventional treatments were similar in both groups (4.57 vs 5.07, P > 0.05). Overall survival was lower in the SC group than the NSC group (P = 0.019), with 1-, 3-, and 5-year survival rates of 82.1% vs 85.1%, 43.2% vs 56.8%, and 27.0% vs 45.2%, respectively. Survival of patients with AJCC stage I disease in the SC group was lower than that of the NSC group (P = 0.029). CONCLUSION: Seroclearance in patients with hepatitis B-related primary liver cancer has protective effects with respect to tumorigenesis, cirrhosis, and portal hypertension but confers worse prognosis, which may be due to the frequent occurrence of highly malignant ICC and CHC.

Propofol inhibits invasion and proliferation of C6 glioma cells by regulating the Ca2+ permeable AMPA receptor-system xc- pathway.

Anesthetics are documented to affect tumors; therefore, we studied the antiglioma effect of propofol on proliferation and invasiveness of glioma cells and explored the underlying mechanism. C6 glioma cells were cultured and treated with propofol, and cell viability, invasiveness, and migration were measured. Glutamate release was measured using an enzyme-catalyzed kinetic reaction. xCT protein and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor GluR2 subunit protein expression was assessed with Western blot analysis and immunofluorescent staining. We observed that propofol significantly inhibited C6 glioma cell viability, invasiveness, and migration and decreased glutamate release. An agonist of the cystine/glutamate antiporter system (system xc-), N-acetylcysteine (NAC), reversed propofol's effects, and propofol could inhibit C6 glioma cell proliferation by adding excess exogenous glutamate (100µM). Finally, propofol increased the surface expression of GluR2, but decreased surface expression of xCT. The effects of propofol on surface expression of GluR2 and xCT could be rescued by (R, S)-AMPA, an agonist of Ca2+ permeable AMPA receptor (CPAR). Thus, propofol can inhibit cell viability, invasiveness, and migration of C6 glioma cells, and the CPAR-system xc- pathway contributes to these events.

Plasma Metabolic Profile Determination in Young ST-segment Elevation Myocardial Infarction Patients with Ischemia and Reperfusion: Ultra-performance Liquid Chromatography and Mass Spectrometry for Pathway Analysis.

BACKGROUND: This study was to establish a disease differentiation model for ST-segment elevation myocardial infarction (STEMI) youth patients experiencing ischemia and reperfusion via ultra-performance liquid chromatography and mass spectrometry (UPLC/MS) platform, which searches for closely related characteristic metabolites and metabolic pathways to evaluate their predictive value in the prognosis after discharge. METHODS: Forty-seven consecutive STEMI patients (23 patients under 45 years of age, referred to here as "youth," and 24 "elderly" patients) and 48 healthy control group members (24 youth, 24 elderly) were registered prospectively. The youth patients were required to provide a second blood draw during a follow-up visit one year after morbidity (n = 22, one lost). Characteristic metabolites and relative metabolic pathways were screened via UPLC/MS platform base on the Kyoto encyclopedia of genes and genomes (KEGG) and Human Metabolome Database. Receiver operating characteristic (ROC) curves were drawn to evaluate the predictive value of characteristic metabolites in the prognosis after discharge. RESULTS: We successfully established an orthogonal partial least squares discriminated analysis model (R2X = 71.2%, R2Y = 79.6%, and Q2 = 55.9%) and screened out 24 ions; the sphingolipid metabolism pathway showed the most drastic change. The ROC curve analysis showed that ceramide [Cer(d18:0/16:0), Cer(t18:0/12:0)] and sphinganine in the sphingolipid pathway have high sensitivity and specificity on the prognosis related to major adverse cardiovascular events after youth patients were discharged. The area under curve (AUC) was 0.671, 0.750, and 0.711, respectively. A follow-up validation one year after morbidity showed corresponding AUC of 0.778, 0.833, and 0.806. CONCLUSIONS: By analyzing the plasma metabolism of myocardial infarction patients, we successfully established a model that can distinguish two different factors simultaneously: pathological conditions and age. Sphingolipid metabolism is the top most altered pathway in young STEMI patients and as such may represent a valuable prognostic factor and potential therapeutic target.

The voltage-gated proton channel Hv1 is expressed in pancreatic islet ß-cells and regulates insulin secretion.

The voltage-gated proton channel Hv1 is a potent acid extruder that participates in the extrusion of the intracellular acid. Here, we showed for the first time, Hv1 is highly expressed in mouse and human pancreatic islet ß-cells, as well as ß-cell lines. Imaging studies demonstrated that Hv1 resides in insulin-containing granules in ß-cells. Knockdown of Hv1 with RNA interference significantly reduces glucose- and K(+)-induced insulin secretion in isolated islets and INS-1 (832/13) ß-cells and has an impairment on glucose- and K(+)-induced intracellular Ca(2+) homeostasis. Our data demonstrated that the expression of Hv1 in pancreatic islet ß-cells regulates insulin secretion through regulating Ca(2+) homeostasis.

The role of interleukin-28b gene polymorphisms in chinese patients with chronic hepatitis C treated with pegylated interferon and ribavirin.

BACKGROUND: Interleukin-28B (IL28B) single nucleotide polymorphism (SNP) rs8099917 has been described to be associated with response to treatment with pegylated interferon and ribavirin (PEG-IFN/RBV) in patients with chronic hepatitis C from the North America, Europe, Asia countries like Japan and Taiwan. Whether this holds true for Chinese patients remains unknown. OBJECTIVES: We aimed to study the effects of IL28B rs8099917 on antiviral therapy responses in Chinese patients with hepatitis C. PATIENTS AND METHODS: IL28B rs8099917 was genotyped in 263 patients with hepatitis C virus (HCV) infection and 244 healthy controls in Tianjin, China using TaqMan SNP genotyping method. The roles of rs8099917 and clinical characteristics in antiviral treatment were analyzed by logistic regression. RESULTS: Among 263 patients with chronic HCV infection, 223 had a TT genotype (84.8%). Frequencies of TG/GG genotypes in patients with hepatitis C were significantly different from those of healthy controls (15.2% vs. 9.0%; P = 0.033). Patients with HCV infection had a higher G allele frequency than healthy controls (7.8% vs. 4.7%; P = 0.044). Univariate analysis revealed no significant association between rs8099917 and sustained virological response (SVR) (P = 0.612). However, it was found that HCV genotypes 2a/3a, age, prothrombin time (PT), albumin (ALB) and cholesterol (CHO) were associated with SVR. In multivariate analysis, only ALB was significantly an independent predictor of SVR (OR = 1.223; 95%CI: 1.046-1.430; P = 0.011). CONCLUSIONS: In contrast with T, rs8099917 G is a susceptible allele to HCV in China. ALB can independently predict SVR. Rs8099917 may play a quiet role to predict treatment response of patients with hepatitis C who received PEG-IFN/RBV therapy in China.

Perinodular ductular reaction/epithelial cell adhesion molecule loss in small hepatic nodules.

AIM: To investigate if loss of epithelial cell adhesion molecule (EpCAM) is associated with microinvasion in hepatocellular carcinomas (HCCs) in the presence of chronic hepatitis B. METHODS: The expression of EpCAM, cytokeratin 7 (CK7) and CK19 in 112 hepatic nodules was studied, including 20 HCCs with nodules ≤ 3 cm, 26 HCCs with nodules > 3 cm, 20 high-grade dysplastic nodules, 26 cirrhotic, large regenerative nodules and 20 cases of cirrhosis. RESULTS: Membranes of ductular reaction (DR) hepatobiliary cells, interlobular bile duct and some hepatic cells were positive for EpCAM expression. Active expression of DR/EpCAM was observed in the majority of noninvasive nodules (50/66, 75.76%); however, expression was absent in the major area of invasion in HCCs (42/46, 91.30%). DR/EpCAM loss in HCCs ≤ 3 cm was higher than in high-grade dysplastic nodules (HGDNs) (P < 0.05), cirrhotic, large regenerative nodules and cirrhosis (P < 0.01). Furthermore, patients (20 HCCs ≤ 3 cm, 26 HCCs > 3 cm, 20 HGDNs) with DR/EpCAM expression had a higher overall survival rate (P < 0.01) and lower early recurrence rate (P < 0.01). DR/EpCAM expression showed a close relationship with DR/CK7 and DR/CK19 expression (P < 0.01). The area under the receiver operating characteristic (ROC) curve of DR/EpCAM was similar to that of DR/CK7 and DR/CK19 (P > 0.05). The diagnostic specificity and diagnostic accuracy were both increased when DR/EpCAM, DR/CK7 and DR/CK19 were combined (P < 0.01). CONCLUSION: DR/EpCAM loss may be a useful marker for determining microinvasion in HCCs ≤ 3 cm, but also for predicting prognosis.

Expression and clinical significance of livin protein in hepatocellular carcinoma.

In this study, the two-step PV method of immunohistochemistry was used to determine livin protein expression in HCC tissues, pericarcinoma tissues, hepatitis/hepatic cirrhosis tissues, and normal hepatic tissues, and livin protein expression was detected in the blood plasma of patients with HCC before and after surgery, subjects with hepatic cirrhosis and hepatitis, and healthy blood donors using ELISA. Livin protein expression was significantly higher in HCC tissues than that in normal hepatic tissues and hepatitis/hepatic cirrhosis tissues, with no significant difference between HCC tissues and pericarcinoma tissues. The HCC patients with positive livin protein expression had a significantly higher survival rate than those with negative livin protein expression. Livin protein expression was significantly higher in the blood plasma of patients with HCC before and after surgery and in patients with hepatic cirrhosis and hepatitis than that in healthy blood donors, whereas livin protein expression in the blood plasma of patients with HCC was not significantly different from that of patients with hepatic cirrhosis and hepatitis. Livin protein expression in HCC tissues did not correlate with that in the blood plasma of the same HCC patients. Livin protein expression may be a potential, effective indicator for assessing prognosis in patients with HCC.

Gradually increased Golgi protein 73 expression in the progression of benign liver diseases to precancerous lesions and hepatocellular carcinoma correlates with prognosis of patients.

AIM: Serum Golgi protein 73 (sGP73) is a novel biomarker for hepatocellular carcinoma (HCC). However, there are few reports on the pattern of GP73 expression in the progression of benign liver diseases to precancerous lesions and HCC. This study aimed to investigate GP73 expression and its correlation with clinicopathological parameters. METHODS: Tissue GP73 (tGP73) levels were detected in specimens of group A (n = 186) including HCC, peritumoral tissue (PTL), high/low-grade hepatic atypical hyperplasia (AH), chronic hepatitis B (CHB) and normal controls (NC) by immunohistochemistry, and GP73 expression in group B (n = 159) and group C (n = 16) were detected by reverse transcription polymerase chain reaction and western blot, respectively. sGP73 levels were detected in subjects of group D (n = 287) by enzyme-linked immunoassay. RESULTS: GP73 expression increased gradually from NC, CHB, PTL to high-grade AH and HCC at both protein and mRNA levels (P < 0.05), while sGP73 in the HCC group was lower than in the liver cirrhosis (LC) group (P < 0.001). Both tGP73 and sGP73 levels were negatively associated with tumor size and tumor-node-metastasis stage, and tGP73 levels were positively associated with tumor differentiation. The high-tGP73 group showed significantly better overall and disease-free survival than the low-tGP73 group (P = 0.008, P = 0.018). Multivariate analysis revealed that the tGP73 level was an independent prognostic factor for HCC, but not sGP73. CONCLUSION: GP73 expression pattern suggests that the regulatory mechanism of GP73 is related to the progression of chronic liver diseases. Furthermore, a high level of tGP73 is a favorable prognostic factor for HCC.

[Diagnostic value of absent ductular reaction at hepatocellular-stromal boundaries in early stage hepatocellular carcinoma].

OBJECTIVE: To investigate the role of absent ductular reaction (DR) at hepatocellular-stromal boundaries in early stage hepatocellular carcinoma (HCC) with cirrhosis in patients with chronic hepatitis B. METHODS: Cytokeratin (CK)7 and CK19 expression was detected by the SP immunohistochemistry method in 112 hepatic nodules taken from 20 cases of early HCC, 26 cases of HCC with nodules more than 3 cm, 20 cases of high-grade dysplastic nodule (HGDN), 26 cases of low-grade dysplastic nodule (LGDN), and 20 cases of cirrhosis (CIR). DR/CK7 and DR/CK19 were assessed separately on a semi-quantitative scale and statistically analyzed. RESULTS: The mean age of the patients in the study was 53.71 years-old, and the study population consisted of 73 males and 39 females. The follow-up time ranged from 3 to 90 months. Positive CK7 and CK19 staining was detected in the cytoplasm of DR-positive hepatobiliary cells, interlobular bile duct, and a portion of hepatic cells. All of the DR/CK7- and DR/CK19-positive cells were localized around the non-invasive nodules. Specimens with focal or diffuse DR/CK7- and DR/CK19-loss had more robust stromal invasion. Specimens from early HCC cases showed greater DR/CK19 loss than specimens from HGDN cases, LGDN cases and CIR cases (all P less than 0.01). DR/CK7 loss of early HCC was less than HCC with nodules more than 3 cm (P less than 0.05), and more than LGDN cases and CIR cases (both P less than 0.01).The area under the receiver operating characteristic curve of DR/CK7 was very similar to that of DR/CK19 (P more than 0.05). Pearson's correlation analysis indicated that DR/CK7 and DR/CK19 were positively correlated with tumor-free time (P less than 0.01) and negatively correlated with early recurrence time as well as death rate (both P less than 0.01). Furthermore, cases showing DR/CK7 or DR/CK19 loss had lower overall survival rate and tumor-free survival rate (P less than 0.01) and higher early recurrence rate (P less than 0.01). CONCLUSION: DR/CK7 and DR/CK19 immunostaining may help to distinguish non-invasive HGDNs from both minimally-invasive and overtly-invasive HCCs by identifying small foci of invasion and predicting increased risk of invasiveness.

Tumor-infiltrating FoxP3+ Tregs and CD8+ T cells affect the prognosis of hepatocellular carcinoma patients.

PURPOSE: Tumor-infiltrating lymphocytes are considered to represent a host immune response against tumor. This study was carried out to analyze the effect of both FoxP3+ regulatory T cells (Tregs) and CD8+ T lymphocytes in prognostic value of hepatocellular carcinoma (HCC) patients. METHODS: Expressions of FoxP3, CD4, CD8 and CD34 in patient-matched tumors and peritumoral tissues were assessed by immunohistochemistry for 54 HCC patients. The prognostic effect of groups with high and low numbers was evaluated by the Kaplan-Meier and Cox model analysis using median values as a cutoff. RESULTS: Compared with the corresponding peritumoral tissue, the density of intratumoral Tregs was significantly higher, while the density of intratumoral CD8+ T cells was lower (p < 0.001 and p = 0.013, respectively). In addition, tumor-infiltrating Tregs were positively correlated with microvessel density in tumors (r = 0.334, p = 0.020). The high intratumoral Tregs density group showed a significantly lower survival rate (overall survival, p = 0.018; disease-free survival, p = 0.029). Multivariate Cox analysis revealed that intratumoral Tregs density was an independent prognostic factor for HCC. CONCLUSIONS: Tumor-infiltrating Tregs may promote HCC progression by fostering angiogenesis and decreasing CD8+ T cells. High tumor-infiltrating Tregs are thought to be an unfavorable prognostic indicator for HCC.

[Analysis of the relationship between hepatitis B virus precore and basal core promoter mutations and acute-on-chronic liver failure].

OBJECTIVE: To analyze the relationship between hepatitis B virus (HBV) precore (PC) and basal core promoter (BCP) mutations and HBV-related acute-on-chronic liver failure (HB-ACLF). METHODS: Forty-four patients with HB-ACLF and 28 patients with chronic hepatitis B (CHB; used as controls) were enrolled and venous blood samples were collected from all individuals. The PC and BCP gene fragments were amplified by nested PCR. HBV genotype and BCP/PC mutations were determined by direct sequencing and analysis by BioEdit (version 7.0.9.0). Ten of the HB-ACLF patients were selected for follow-up (range: 2-8 weeks), which included once weekly sera collection to determine the relation of mutations and treatment response. Serum levels of HBV DNA were measured by real-time PCR assay, and alanine aminotransferase, total bilirubin, creatinine and albumin were measured by standard biochemical assay and used to determine the MELD score. RESULTS: All 44 HB-ACLF patients were infected with HBV genotype C. In the CHB group, 26 patients were infected with genotype C and two with genotype B. Single mutations (A1762T, G1764A, T1753V, G1896A, and G1899A) and combined mutations (A1762T + G1764A, G1896A + G1899A, T1753V+ A1762T + G1764A, G1896A + G1899A + A1762T + G1764A, and A1762T + G1764A + G1896A) were more frequently detected in HB-ACLF patients than in CHB patients (P less than 0.05). A significantly higher proportion of PC/BCP wild-type sequences was found in patients with CHB than in patients with HB-ACLF (17.9% vs. 2.3%; x² = 5.440, P = 0.020). The proportion of patients carrying both PC and BCP mutations was significantly higher in HB-ACLF patients than in CHB patients (79.6% vs. 39.3%; x² = 12.021, P = 0.001). The proportion of patients carrying only BCP mutation was 42.9% in the CHB group and 20.5% in the HB-ACLF group (x² = 4.157, P = 0.041). No occurrences of only PC mutation were detected in either the CHB or HB-ACLF group. The combined mutations were present in all 10 of the HB-ACLF follow-up patients. Mutations G1899A, T1753V, and A1846T were correlated with disease recovery. Significant decreases in the MELD score were accompanied by decreases in the A1846T mutation. CONCLUSION: Significantly more HB-ACLF patients carried HBV with mutations in the PC and BCP than CHB patients. Moreover, more HB-ACLF patients carried HBV with PC + BCP combined mutations and PC mutation only. The G1899A, T1753C, and A1846T mutations were associated with HB-ACLF response to treatment and improvement in liver function.

Aberrant methylation of SPARC in human hepatocellular carcinoma and its clinical implication.

AIM: To investigate the methylation status of secreted protein acidic and rich in cysteine (SPARC) in human hepatocellular carcinoma (HCC) and evaluate its clinical implication. METHODS: The methylation status of SPARC was analyzed in one HCC cell line (SMMC-7721) and 60 pairs of HCC and corresponding nontumorous tissues by methylation-specific polymerase chain reaction and bisulfite sequencing. The expression of SPARC mRNA and protein were examined by reverse transcription polymerase chain reaction and immunohistochemistry, respectively. The correlations between the methylation status and the gene expression, the clinicopathological parameters, as well as the prognosis after surgery were analyzed. RESULTS: In the SMMC-7721 cell line, the loss of SPARC expression was correlated with the aberrant methylation and could be reactivated by the demethylating agent 5-aza-2'-deoxycytidine. Methylation frequency of SPARC in HCC was significantly higher than that in the corresponding nontumorous tissues (45/60 vs 7/60, P < 0.001), and it was correlated with the pathological classification (P = 0.019). The downregulation of the SPARC mRNA expression in HCC was correlated with the SPARC methylation (P = 0.040). The patients with methylated SPARC had a poorer overall survival than those without methylated SPARC (28.0 mo vs 41.0 mo, P = 0.043). CONCLUSION: Aberrant methylation is an important mechanism for SPARC inactivation in HCC and SPARC methylation may be a promising biomarker for the diagnosis and prognosis of HCC.

[Tumor-infiltrating FoxP3+ Tregs are associated with CD34 expression and prognosis of hepatocellular carcinoma].

OBJECTIVE: To investigate the correlation between FoxP3+ regulatory T lymphocytes (Tregs) in hepatocellular carcinomas (HCCs) and peritumoral tissues with CD34 expression and patient prognosis. METHODS: Fifty-five sets of patient-matched tumors and peritumoral tissues were obtained during curative resection for HCC. In situ immunohistochemistry was used to assess and comparatively analyze Treg presence and CD34 expression in each specimen set. The relation between quantified Tregs values and various clinicopathologic factors were evaluated by the Spearman Rank Correlation test. Univariate (Log Rank test) and multivariate (Cox Regression model) analyses were used to determine the potential prognostic value of each factor. RESULTS: The average number of intratumoral Tregs was significantly higher than that in corresponding peritumoral tissues (10.8 (range: 4.4 to 19.4) vs. 1.4 (0.6 to 3.2), respectively; P less than 0.01). The presence of intratumoral Tregs correlated with up-regulated CD34 expression (r = 0.279, P less than 0.05). Increased number of intratumoral Tregs were significantly associated with decreased rates of overall survival (OS, P less than 0.05) and disease-free survival (DFS, P less than 0.05), and was identified as an independent prognostic factor (OS, hazard ratio (HR) = 3.310, 95% confidence interval (CI): 1.368-8.007, P less than 0.01; DFS, HR = 2.666, 95% CI: 1.321 to 6.394, P less than 0.01). CONCLUSION: Intratumoral infiltration by Tregs is a marker of poor prognosis in HCC patients.

[Studies on the relationship between polymorphism of IL-28B rs8099917 and the outcome of HBV infection].

OBJECTIVE: To investigate the correlation between IL-28B rs8099917 polymorphism and the outcome of HBV infection. METHODS: Genotype of rs8099917 (T > G) in IL-28B locus was determined by TaqMan SNP genotyping from 486 individuals which including 199 chronic HBV carriers (including 100 HBV-induced liver cirrhosis and 99 HBV-related HCC). 143 people with self-limited infection and 144 healthy people served as controls. Multivariate analysis was used to assess the effect of IL-28B rs8099917 SNP among all the studied groups. RESULTS: Distribution of genotype and allele of the rs8099917 locus were in accordance with Hardy-Weinberg equilibrium in different groups or with the total population. The frequencies of the rs8099917 TT, GT, GG genotypes were 89.3%, 10.5% and 0.2%, and the frequency of allele T and G accounted for 94.5% and 5.5%, respectively. In respect of genotype or allele frequency, there was no significant differences found among the groups (P > 0.05). When comparing with the TT genotype, data from the multinomial logistic analysis showed that the ORs and (95%CI) of TG/GG genotypes were 1.589 (0.735 - 3.437), 1.351 (0.550 - 3.316) and 1.704 (0.717 - 4.052), respectively. The genotype frequencies in different groups with different clinical features showed that TG/GG genotypes significantly increased the risk of r-GTII(+) for individuals with HBV-related HCC (χ(2) = 17.534, P = 0.001), with OR as 14.821 (3.227 - 68.064). It was particularly so for males (χ(2) = 14.924, P = 0.014), with OR (95%CI) as 45.000 (2.772 - 730.571). CONCLUSION: IL-28B rs8099917 SNP had no correlation with the outcome of HBV infection.

Enhanced specificity of real-time PCR for measurement of hepatitis B virus cccDNA using restriction endonuclease and plasmid-safe ATP-dependent DNase and selective primers.

The persistence of covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) in hepatocytes plays a key role in viral persistence and resistance to therapy. Therefore, quantitative cccDNA measurement is of clinical importance for evaluating the efficacy of antiviral drugs, selecting an appropriate treatment strategy, and predicting the prognosis. Current established methods for measurement of cccDNA need further improvement. A modified method was developed using digestion with restriction endonucleases that do not recognize sites in the HBV DNA and plasmid-safe ATP-dependent DNase (PSAD), and using a cccDNA-specific primer set in a real-time PCR reaction. The cccDNA-specific primer has a similar amplification efficiency as a commercial kit. Treatment of samples with restriction endonuclease followed by PSAD digestion increased significantly the specificity of a cccDNA-selective primer set compared with other treatments (P<0.05). Analysis of 35 serum and liver DNA samples from patients with hepatocellular carcinoma demonstrated that the amount of serum cccDNA is beyond the minimum detection limit and that the liver cccDNA quantity is about 0-49.2 copies/cell, consistent with previous reports. Taken together, this method has the potential for evaluating the efficacy of antiviral drugs.

[Resistance mutation patterns of hepatitis B virus in patients with suboptimal response to adefovir dipivoxil therapy after lamivudine resistance].

OBJECTIVE: To investigate the resistance mutation patterns of hepatitis B virus(HBV) during adefovir dipivoxil (ADV) monotherapy or combination therapy after lamivudine(LAM) resistance. METHODS: Serum samples from fifteen patients with suboptimal viral response to ADV therapy after LAM resistance were collected. The RT region of HBV P gene was PCR-applied, cloned and sequenced, and the mutation patterns in relation to resistance were analyzed. RESULTS: The ADV resistance mutation patterns of A181T+N236T, A181V, A181T were selected in ADV monotherapy group. The LAM resistance mutation patterns of M204V+L180M, M204V+L180M+L229V, M204I+L80I, M204V+L180M+V207I were detected in the combination therapy group. 20% of clones from three serum samples were detected double resistance to LAM and entecavir (ETV) in the combination therapy group, the resistance patterns were M204I+L80I+T184I (2/10), M204V+L180M+T184S (2/10), and M204V+L180M+G173L+S202G (2/10) respectively. I269L clones were detected in two serum samples from both two groups and P109S clones also detected in the one from monotherapy group. CONCLUSIONS: In the patients with suboptimal viral response to ADV therapy after LAM resistance, the ADV resistance mutation patterns of A181T+N236T, A181V and A181T could easily be selected during ADV monotherapy; while in the patients with combination therapy, the LAM resistance mutation patterns of M204V+L180M, M204V+L180M+L229V, M204I+L80I, and M204V+L180M+V207I were predominant, the ETV resistance mutation T184I/S and S202G could be selected. The mutation patterns of I269L and P109S may impact the responses to ADV therapy in some patients.

Identification and validation of specific methylation profile in bile for differential diagnosis of malignant biliary stricture.

OBJECTIVE: This study was aimed to identify the specific methylation profile in bile specimens of pancreaticobillary diseases for differential diagnosis of malignant biliary stricture. DESIGN AND METHODS: In a total of 80 bile specimens from pancreaticobillary diseases, the methylation status of 19 tumor suppressor genes were analyzed by methylation-specific PCR and the methylation index (MI) were compared between the malignant and benign groups. RESULTS: Methylation of DKK3, p16, SFRP2, DKK2, NPTX2 and ppENK were more frequently detected in the bile of malignant biliary strictures than benign patients. When setting MI 0.5 as the threshold, this 6-gene panel could distinguish the malignant biliary stricture with a high sensitivity, specificity and accuracy (77.27%, 77.78% and 77.50%, respectively). CONCLUSION: The methylation profile including 6 specific genes in bile may be a promising biomarker for differential diagnosis between malignant and benign biliary strictures.